Microbial method for producing lignin peroxidase

ABSTRACT

Method for producing lignin-peroxydase from the fungus Phanerochaete chrysosporium. The method comprises a first step of culture wherein the phospholipids and the emulsified fatty acids are added to the culture medium; a second step during which veratrylic alcohol is added to the culture medium, the culture medium being partially renewed for the second culture step and totally renewed for the third and fourth culture steps, by varying the content of constituents in said medium. Application to the production of lignin-peroxydase with important yields.

The present invention relates to an improved method of producingligninolytic enzyme or lignin peroxidase.

BACKGROUND OF THE INVENTION

In SCIENCE, (1983) 221, pp. 661-663, MING TIEN and T. KENT KIRK havedescribed an extracellular enzyme produced by Phanerochaetechrysosporium Burdsall, which is a basidiomycetes fungus; in thepresence of hydrogen peroxide, this enzyme is capable of causing theoxidizing degradation of various model compounds having ligninsubstructures, as well as the degradation of fir or birch lignin.

This enzyme, called "lignin peroxidase", has been characterized [M. TIENand T. K. KIRK, PROC. NATL. ACAD. SCI. USA, (1984) vol. 81, pp.2280-2284] as being a glycoprotein of about 42 kDa.

French patent 2 574 427 claims two novel strains of Phanerochaetechrysosporium Burdsall which are capable of developing a particularlyhigh ligninolytic activity in media of unlimited nitrogen content,whereas the previously known strains produced the ligninolytic enzymeonly in a nitrogen-deficient culture medium, the biodegradation processwhich produced this enzyme also being very slow under these conditions.

According to said patent, the culture medium suitable for promoting theproduction of lignin peroxidase contains a source of assimilablenitrogen which can be asparagine, ammonium nitrate or ammonium tartrate;it also contains a source of assimilable carbon such as glucose,mannose, starch, melibiose, mannitol, xylose, maltose, adonitol,arabitol, fructose, sorbitol, raffinose, xylitol, D(+)-trehalose orglycerol, the last of these being preferred; it further contains asource of assimilable mineral salts such as iron citrate, KH₂ PO₄,ZnSO₄, MnSO₄, CaCl₂, CuSO₄, NaCl, FeSO₄, CoSO₄, AlK(SO₄)₂, H₃ BO₃, Na₂MoO₄ or MgSO₄.

French patent 2 600 077 proposes significantly increasing the productionyields of lignin peroxidase by cultivating the fungus Phanerochaetechrysosporium on a base culture medium supplemented with a stimulantselected from unsaturated fatty acids, natural amino acids and mixturesthereof. The preferred unsaturated fatty acids are oleic acid, linoleicacid, palmitoleic acid and arachidonic acid, preferably in emulsifiedform, and the preferred natural amino acids are serine, threonine,isoleucine, glycine, valine and tyrosine, but more particularlyisoleucine, serine, threonine and glycine. The optimum concentration ofoleic acid is about 800 mg/liter or, in the case of emulsified oleicacid, about 400 mg/liter; the optimum concentration of isoleucine isabout 6.5 mg/liter.

It should be pointed out that JAGER et al. (APPLIED AND ENVIRONMENTALMICROBIOLOGY, Nov. 1985, pp. 1274-1278) have proposed adding adetergent, such as TWEEN 80 or TWEEN 20, to the culture medium of thefungus, in submerged cultures, in order to increase the production oflignin peroxidase in proportions comparable to that currently obtainedin stationary cultures. FAISON & KIRK [APPL. ENVIRON. MICROBIOL., (1985)49, pp. 299-304] and LEISOLA et al. [J. BIOTECHNOL., (1985) 3, pp.97-107] have reported that the addition of veratryl alcohol, which is asecondary metabolite of Phanerochaete chrysosporium, increases thesynthesis of lignin peroxidase.

In an article published in ENZ. MICROBIAL TECHNOL., (1987) 9, pp.245-249, ASTHER et al. have shown that, in the presence of exogenousoleic acid emulsified by TWEEN 80, there is a significant increase inthe production of lignin peroxidase and a considerable reduction in thefermentation time required to reach the maximum enzymic activity.

In a more recent article, published in APPL. MICROBIOL. BIOTECHNOL.,(1988) 27, pp. 393-398, ASTHER et al. have reported that the productionof lignin peroxidase by the fungus Phanerochaete chrysosporium isfurther increased when olive oil supplemented with soya azolectin, whichconstitutes a source of phospholipids, is added to the culture medium.

In a communication delivered at the Annual Meeting of the ASM which tookplace from May 8 to 13, 1988 in Miami, Fla., United States, ASTHER etal. reported that a maximum production of lignin peroxidase is obtainedfrom cultures of Phanerochaete chrysosporium when culture is carried outat two different temperatures, namely at 37° C. during the growth phaseof the mycelium, for the first two days of incubation, and then at 30°C. during the lignin peroxidase production phase.

LEISOLA et al. [q.v. the above-cited article published in J. BIOTECHNOL.in 1985] have identified four hemes containing lignin peroxidaseactivity in extracellular fluid of three-day-old cultures ofPhanerochaete chrysosporium BKM-F-1767, induced by veratryl alcohol.JAGER et al. [APPL. ENVIRON. MICROBIOL., (1985) 50, pp. 1274-1278] haveseparated 8 peaks with an absorbance at 409 nm (hemoproteins), assumingthat relatively minor differences between the profiles of the proteinssimply reflect the age of the cultures.

OBJECTS OF THE INVENTION

The aim of the present invention was to provide a method of producinglignin peroxidase from Phanerochaete chrysosporium under controlledtemperature conditions which are different during the growth phase ofthe mycelium and during the actual lignin peroxidase production phase,and which make it possible to favour the production of the hemoproteinshaving the maximum lignin peroxidase activity, at the expense of biomassproduction.

SUMMARY OF THE INVENTION

The present invention relates to a method of producing lignin peroxidasefrom a fungus known as Phanerochaete chrysosporium, in a culture mediumcontaining activators of the enzyme, such as phospholipids and veratrylalcohol, which method is characterized in that it comprises:

a first step involving the culture of cells of Phanerochaetechrysosporium for an incubation period of about 2 days, in a syntheticculture medium comprising salts of potassium, calcium and magnesium,trace elements (iron, zinc, manganese, copper), an appropriate source ofnitrogen, a source of carbon advantageously consisting of glycerol,yeast extract, a source of phospholipids and a source of emulsifiedfatty acids;

a second step involving culture of the mycelium formed during the firststep, for a period of about 3 days (from D3 to D5), in a culture mediumwhich has been partially renewed (advantageously to the extent of about15%), to which veratryl alcohol--an activator and protector of ligninperoxidase production--has been added, and whose content ofphospholipids has been reduced to about 1/7-1/8 of what it was in theculture medium of the first step, the present culture medium notcontaining emulsified fatty acids;

a third step involving culture at the optimum lignin peroxidaseproduction, i.e. after about 5 days, during which the whole of theculture medium of the second step is replaced with a synthetic culturemedium analogous to that of the first step, which contains the sameproportion of phospholipids and veratryl alcohol as the culture mediumof the second step, and in which the yeast extract, the source ofnitrogen and the source of carbon--advantageously consisting ofglycerol--have been reduced to 1/4 of their content in the culturemedium of the first step, these three components together forming apartial regeneration medium for the biomass;

a fourth step consisting in continuing the culture withnon-proliferating cells for about 8 days (from D6 to D13), the culturemedium being totally renewed every 24 hours and replaced with a mediumnot containing yeast extract, emulsified fatty acids or glycerol, butcontaining the activators and protectors of the enzyme production,consisting of veratryl alcohol and phospholipids, in the sameproportions as the culture media of the second and third steps; and

a fifth step involving separation of the enzyme produced and, ifnecessary, purification of said enzyme.

The precise strains of the white rot fungus Phanerochaete chrysosporiumBurdsall employed in this invention have been deposited under theBudapest Treaty at the Collection Nationale de Cultures deMicroorganisms (CNCM) at the Pasteur Institute, 25 Rue de Dr. Roux,Paris 75015 France, and are available to the public. These strains ofPhanerochaete chrysosporium Burdsall bear the identifying numbers CNCMI-398 and CNCM I-399. These same strains and methods for producing themare also described and claimed in European Patent No. 188,931. Anotherdesignation at the CNCM, Institute Pasteur, Paris for CNCM I-398 isINA-12, as disclosed by Applicants in Enzyme Microbial Technology, Vol.13, September 1991 at pp. 727-733.

In one advantageous mode of carrying out the method of producing ligninperoxidase according to the present invention, the first step involvingculture of the cells takes place at an incubation temperature of about37° C., whereas the next steps are carried out at incubationtemperatures of about 30° C.

In another advantageous mode of carrying out the method according to thepresent invention, the first step involving culture of the cells takesplace in a culture medium buffered at pH 6.5, whereas the next steps arecarried out in a culture medium buffered at pH 5.5.

In yet another advantageous mode of carrying out the method of producinglignin peroxidase according to the present invention, the source ofphospholipids advantageously consists of soya azolectin.

In another advantageous mode of carrying out the method of producinglignin peroxidase according to the present invention, the culture mediumof the first step has a content of nitrogen source of the order of 1.84g/liter for a content of carbon source (glycerol) of the order of 10g/liter, for a content of phospholipids (soya azolectin) of the order of0.75 g/liter and for a content of yeast extract of 1 g/liter.

In yet another advantageous mode of carrying out the method of producinglignin peroxidase according to the present invention, the culture mediumof the second step differs from the culture medium of the first step byits pH (5.5), its reduced content of phospholipids (of the order of 0.1g/liter) and its content of veratryl alcohol (of the order of 2.5 mM).

In another advantageous mode of carrying out the method of producinglignin peroxidase according to the present invention, the culture mediumof the third step differs from the culture medium of the second step byits reduced content of glycerol (about 2.5 g/liter), nitrogen source (inparticular about 0.46 g/liter of diammonium tartrate) and yeast extract(0.25 g/liter).

In another advantageous mode of carrying out the method of producinglignin peroxidase according to the present invention, the culture mediumof the fourth step differs from the culture medium of the third step inthat it does not contain yeast extract, a source of nitrogen or a sourceof carbon.

In another advantageous mode of carrying out the method of producinglignin peroxidase according to the invention, the third and fourth stepsare carried out continuously.

The method according to the present invention affords a considerableincrease in the lignin peroxidase productivity and the lignin peroxidaseactivity compared with all the methods known hitherto. In fact, whereasa lignin peroxidase activity of the order of 22.4 nKat.ml⁻¹ is obtainedin the case where a culture medium of Phanerochaete chrysosporium issupplemented with oleic acid emulsified with Tween 80 at a concentrationof 0.04% (weight/volume), and whereas the lignin peroxidase activity isof the order of 33.3 nKat.ml¹ when the culture medium is supplementedwith azolectin as the source of phospholipids and olive oil as thesource of lipids, the lignin peroxidase activity can reach 40.6nKat.ml⁻¹ when using the method according to the present invention, inwhich culture media supplemented successively with phospholipids andemulsified fatty acids, and then with veratryl alcohol andphospholipids, are used at different incubation temperatures of 37° C.for the first culture medium and 30° C. for the second. Moreover,whereas the lignin peroxidase productivity is of the order of 40.6nKat.ml⁻¹.day⁻¹ when using the method according to the invention, it isas low as 4.5 nKat.ml⁻¹.day⁻¹ when the culture medium contains oleicacid emulsified with Tween 80, it is 8.3 nKat.ml⁻¹.d⁻¹ when the culturemedium contains azolectin and olive oil and it is 1.6 nKat.ml⁻¹.day⁻¹when the culture medium contains veratryl alcohol 1 mM, with anincubation temperature of 39° C., as described in the prior art cited.

The FPLC (fast protein liquid chromatography) profiles of extracellularproteins obtained from cultures incubated at 37° C. for the first twodays and then at 30° C., with the changes of culture media according tothe present invention, show the presence of several hemoproteins, nineof which have lignin peroxidase activity, with a large peak n° 4.

In addition to the foregoing provisions, the invention also includesother provisions which will become apparent from the followingdescription.

PREFERRED EMBODIMENTS OF THE INVENTION

The invention will be understood more clearly with the help of thefollowing additional description referring to the attached drawings, inwhich:

FIG. 1 shows the flow diagram of the method according to the presentinvention, and

FIG. 2 shows the FPLC profiles of the isoenzymes of lignin peroxidaseafter 5 days of culture, as a function of the culture conditions.

It must be clearly understood, however, that these drawings and thecorresponding descriptive sections are given solely in order toillustrate the subject of the invention, without in any way implying alimitation.

EXAMPLE 1 OF LIGNIN PEROXIDASE PRODUCTION

The microorganism used is the strain Phanerochaete chrysosporium INA-12(CNCM n° I-398).

Cells of Phanerochaete chrysosporium INA-12 immobilized on polyurethanefoam are treated in the following manner:

1. 6×10⁷ conidiospores are inoculated into a culture medium of thefollowing composition:

    ______________________________________                                        Solution of salts                                                             Potassium dihydrogen phosphate KH.sub.2 PO.sub.4                                                       2       g/l                                          Calcium chloride CaCl.sub.2.2H.sub.2 O                                                                 0.14    g/l                                          Magnesium sulphate MgSO.sub.4.7H.sub.2 O                                                               0.7     g/l                                          Solution of trace elements                                                    Iron (ferrous) sulphate FeSO.sub.4.7H.sub.2 O                                                          0.07    g/l                                          Zinc sulphate ZnSO.sub.4.7H.sub.2 O                                                                    0.0462  g/l                                          Manganese sulphate MnSO.sub.4.H.sub.2 O                                                                0.035   g/l                                          Copper sulphate CuSO.sub.4.5H.sub.2 O                                                                  0.007   g/l                                          Solution of vitamins                                                          Thiamine hydrochloride (vitamin B.sub.1)                                                               0.0025  g/l                                          Source of nitrogen                                                            Ammonium tartrate        1.84    g/l                                          Source of carbon                                                              Glycerol                 10      g/l                                          Yeast extract            1       g/l                                          Soya azolectin (phospholipids)                                                                         0.75    g/l                                          Oleic acid emulsified with Tween 80                                                                    0.4     g/l                                          pH of the culture: 6.5 (the buffer used is                                    2,2-dimethylsuccinic acid)                                                    ______________________________________                                    

INTERMEDIATE TEMPERATE: 37° C.

After 100% O₂ has been blown in for two minutes, culture is carried out,without agitation, in 150 ml Erlenmeyer flasks containing 30 ml of theabovementioned aqueous culture medium, from D0 to D2 at 37° C.

2. 15% of the above-mentioned total medium is withdrawn and replacedwith a culture medium such as described in 1. above, except as regardsthe yeast extract, the soya azolectin and the emulsified oleic acid, thefirst and third of which are omitted, as are the glycerol, thediammonium tartrate, the salts, the trace elements and the vitamins,while the content of soya azolectin in the medium is reduced by a factorof 7.5 (0.1 g/liter) and veratryl alcohol (2.5 mM) is added to saidmedium, the veratryl alcohol and the soya azolectin acting as inductorsof lignin peroxidase production. Culture is continued from D3 to D5inclusive at an incubation temperature of 30° C., which is the optimumtemperature for lignin peroxidase production.

3. On D5, the whole of the culture medium is withdrawn and replaced witha culture medium of the following composition:

    ______________________________________                                        Source of nitrogen                                                            Diammonium tartrate   0.46   g/l                                              Source of carbon                                                              Glycerol              2.5    g/l                                              Yeast extract         0.25   g/l                                              Soya azolectin        0.1    g/l                                              Veratryl alcohol      2.5    mM                                               ______________________________________                                    

The veratryl alcohol and the soya azolectin present in this medium actas inductors and protectors of the lignin peroxidase, as in the culturemedium used in step 2. above, and the glycerol, the ammonium tartrateand the yeast extract together form a regeneration medium for thebiomass. The pH of the culture medium is adjusted to 5.5 and theincubation temperature is 30° C. The glycerol, the source of nitrogenand the yeast extract act on the production of biomass and not on theproduction of enzyme, which is why they are present in the regenerationmedium. However, their concentrations are reduced to 25% of what theyare in the culture medium of step 1. above, so as to limit theproduction of biomass in proportions which do not jeopardize productionof the enzyme.

4. From D6, the whole of the culture medium is withdrawn every 24 hoursand replaced with a culture medium of the same composition as that usedin step 2. above, and culture is continued up to D13 at 30° C. and at pH5.5.

FIG. 1 attached represents a diagram of the method of producing ligninperoxidase by using non-proliferating cells of Phanerochaetechrysosporium, according to the present invention.

In this diagram:

    ______________________________________                                         ##STR1##                                                                             represents the temperature profile,                                    ##STR2##                                                                             represents the replacement of 15% of the culture medium with a               culture medium comtaining 0.1 g/l of azolectin and 2.5 mM veratryl            alcohol,                                                                ##STR3##                                                                             represents the replacement of 100% of the culture medium with a              culture medium containing azolectin (0.1 g/l), veratryl alcohol               (2.5 mM) and a partial regeneration medium for the biomass                    (glycerol 2.5 g/l, ammonium tartrate 0.46 g/l, yeast extract 0.25             g/l), and                                                               ##STR4##                                                                             represents the replacement (every 24 hours) of 100% of the                   culture medium with a culture medium containing azolectin (0.1                g/l) and veratryl alcohol (2.5 mM).                                    ______________________________________                                    

5. The cultures obtained are harvested and filtered on a glass fibrefilter. The supernatant is dialyzed overnight against distilled water.The extracellular medium is then concentrated to 1/10 of its initialvolume by ultrafiltration through an Amicon YM10 membrane.

The proteins contained in the concentrated fluid are determined by FPLC(fast protein liquid chromatography) using a PHARMACIA LCC 500chromatograph equipped with a Mono QHR 515 anion exchange column, with agradient of NaCl in 10 mM sodium cacodylate (pH 5.9). The FPLC profilesof the isoenzymes of lignin peroxidase obtained are shown in FIG. 2attached, in which

FIG. 2A represents the profiles of extracellular proteins ofPhanerochaete chrysosporium INA-12 under standard culture conditionswithout renewal of the culture medium, the incubation temperature being37° C.;

FIG. 2B represents said protein profiles obtained with renewal of theatmosphere (100% oxygen) after two days of culture;

FIG. 2C represents said protein profiles obtained with renewal of theatmosphere with oxygen and changing of 15% of the medium with 2.5 mMveratryl alcohol after two days of culture;

FIG. 2D represents said protein profiles obtained with renewal of theatmosphere with oxygen and changing of 15% of the medium with 2.5 mMveratryl alcohol and 0.1 g/l of azolectin after two days of culture; and

FIG. 2E represents said protein profiles obtained with renewal of theatmosphere with oxygen and changing of 15% of the medium with 2.5 mMveratryl alcohol and 0.1 g/l of azolectin, and changing of theincubation temperature from 37° C. to 30° C. after two days of culture.The absorbance at 405 nm is represented by solid lines and theabsorbance at 280 nm by broken lines; the sloping line represents thegradient of NaCl.

These profiles show that the extracellular fluid contains severalhemoproteins, six of which have lignin peroxidase activity; thesehemoproteins correspond to peaks 1, 2, 3, 4, 5, 6, 7, 8 and 9. 80% ofthe lignin peroxidase activity is associated with peaks 7, 8 and 9. Thecultures whose incubation temperatures were successively 37° C. and then30° C., and those whose incubation temperature was 30° C. only, show anincrease in the proportion of peak 4.

EXAMPLE 2 OF LIGNIN PEROXIDASE PRODUCTION

The conditions of Example 1 are repeated, 100 ml of medium beingintroduced into a 250 ml Erlenmeyer flask; an enzyme production of 46.4nKat/ml is obtained.

EXAMPLE 3 OF LIGNIN PEROXIDASE PRODUCTION

The microorganism used is also the strain Phanerochaete chrysosporiumINA-12 (CNCM n° I-398).

Cells of Phanerochaete chrysosporium INA-12 immobilized on polyurethanefoam are treated as in step 1 of Example 1.

The reactor is inoculated with 2.10⁵ spores/ml. There is directimmobilization in situ in the reactor.

The medium is fed continuously with oxygen introduced through a centralshaft, where the agitating turbine (200 rpm) is also situated. The O₂concentration is regulated to about 60% of saturation of the medium withair. The cells of Phanerochaete chrysosporium immobilized on thepolyurethane foam are placed at the periphery of the bioreactor, aroundthe central shaft.

Steps 2 to 5 are identical to those of Example 1.

The enzyme titre obtained reaches 25 nKat/ml/day.

The method according to the present invention makes it possible tocontrol the production of lignin peroxidase and consequently affords thepossibility of producing this enzyme on an industrial scale.

As is apparent from the foregoing description, the invention is in noway limited to those modes of execution, embodiments and modes ofapplication which have now been described more explicitly; on thecontrary, it encompasses all the variants thereof which may occur tothose skilled in the art, without deviating from the framework or thescope of the present invention.

We claim:
 1. A method for producing lignin peroxidase from a strain of fungus known as Phanerochaete chrysosporium CNCM I-398 and 399, in a culture medium containing activators and protectors of the enzyme, chosen from the group consisting of phospholipids and veratryl alcohol and mixtures thereof comprising the steps of:a) culturing cells of Phanerochaete chrysosporium for an incubation period of about two days, in a synthetic culture medium comprising salts of potassium, calcium and magnesium, and trace elements selected from the group consisting of iron, zinc, magnesium, copper and mixtures thereof, an appropriate source of nitrogen, an appropriate source of carbon, a source of phospholipids, and a source of emulsified fatty acids, which first step takes place at an incubation temperature of about 37° C.±0.5° C., the culture medium being buffered at about pH 6.5; b) culturing the mycelium formed during step a), for a period of about three more days, in a culture medium which has been partially renewed by adding veratryl alcohol together with phospholipids, and whose content of phospholipids has been reduced to 1/7-1/8 of what it was in the culture medium of step a), which present culture medium free from emulsified fatty acids and is buffered at about pH 5.5, at an incubation temperature of about 30° C.±0.5° C.; c) replacing the culture medium of step b) with a synthetic culture medium analogous to that of the step a) having salts, a source of nitrogen, a source of carbon, a source of phospholipids and a source of emulsified fatty acids having the same proportion of phospholipids and veratryl alcohol as the culture medium of step b), and in which the yeast extract, the source of nitrogen and the source of carbon have been reduced to 1/4 of their content in the culture medium of step a), these three components together forming a partial regeneration medium, the incubation temperature being about 30° C.±0.5° C. and the pH of the culture medium being about 5.5; d) continuing the culture with non-proliferating cells for eight days, the culture medium being totally renewed every day and replaced with a medium free of yeast extract, emulsified fatty acids, and glycerol, but containing the activators and protectors of the enzyme production, comprising veratryl alcohol and phospholipids, in the same proportions as the culture media of steps b) and c), the pH and the incubation temperature being the same as during steps b) and c); e) separating the enzyme produced and, f) purifying said enzyme.
 2. A method according to claim 1, wherein the source of phospholipids comprises soya azolectin.
 3. A method according to claim 1, wherein the culture medium of step a) has a content of nitrogen source of about 1.84 g/liter, a content of carbon of about 10 g/liter, a content of phospholipids of about 0.75 g/liter, and a content of yeast extract of about 1 g/liter.
 4. A method according to claim 1, wherein the culture medium of step b) differs from the culture medium of step a) by its lower pH, its reduced content of phospholipids, and its content of veratryl alcohol.
 5. A method according to claim 1, wherein the culture medium of step c) differs from the culture medium of step b) by its reduced content of glycerol, nitrogen source, and yeast extract.
 6. A method according to claim 1, wherein the culture medium of step d) differs from the culture medium of step c) in that it is free from yeast extract, a source of nitrogen, and a source of carbon.
 7. A method according to claim 1, wherein the cultures obtained are harvested and separated by filtration, after which the extracellular medium obtained is concentrated in order to collect the desired lignin peroxidase.
 8. The method according to claim 3, wherein the source of carbon is glycerol.
 9. The method according to claim 3, wherein the source of phospholipid is soya azolectine.
 10. The method according to claim 1, wherein the renewal of the culture medium in step b) is about 15 percent. 